The ability to understand how brains generate behavior both in normal and pathological situations relies on our understanding of the neural computations carried out by behavioral circuits. The nature of chemical communication between neurons is determined by the specific neurotransmitters released onto postsynaptic targets and it was thought for many years that neurons released a single type of transmitter onto all their targets. In recent years, however, it has become clear that key neurons in vertebrate and invertebrate circuits involved in addiction, memory formation, feeding behavior and reproduction contain neurons that violate this rule, releasing multiple neurotransmitters. In this proposal we develop genetic tools to allow mapping of the distribution of specific neurotransmitter release sites in single neurons in multiple colors by modifying the endogenous genetic loci of vesicular transporters for neurotransmitter with fluorescent proteins. This allows both accurate and complete accounting of transmitter release sites since the marker's generation and turnover rely on processes that control the presence of the endogenous protein. Single cell resolution is obtained via an intersectional version of this strategy in which split fluorescent proteins become reconstituted only in specific cells. There is no current technique in any system which can provide this level of resolution. This technology will provide the ability to determine, in complex neurons releasing several chemical substances, the spatial distribution of each of the chemicals and its relationship to downstream targets of that neuron. It will also allow the mapping of temporal changes, either developmental or plasticity-induced, in neurotransmitter release. The technique is developed initially for use in Drosophila, a model organism which has been immensely important for our understanding of both the genetic and circuit basis of behavior, but as a general strategy can also be adapted for use in mammalian brain.